Dual distal insertions drive pimarane-to-miltiradiene rearrangement with enhanced enzymatic activity.

Miltiradiene, the major biosynthetic precursor of abietane diterpenoid natural products, has downstream metabolic derivatives with a significant potential for pharmacologically development. The formation of the characteristic abietane skeleton is attained through the rearrangement of the C13 methyl group in the pimarane intermediate, but the key enzyme mechanism responsible for this rearrangement remains elusive. Previous studies have shown that IrKSL3a in Isodon rubescens generates the pimarane diterpene isopimaradiene, and the insertion of two amino acids in the flexible region distal to the enzyme active center can change the enzyme's function to produce the abietane diterpene miltiradiene. In this paper, mutation studies were conducted on another isopimaradiene synthase IrKSL6 in Isodon rubescens and the miltiradiene synthase SmKSL1 in Salvia miltiorrhiza at these two sites. It was found that the insertion at these two sites is conservative in changing the enzyme function, and it can also affect the solubility of SmKSL1 and its affinity for the substrate. The titer of miltiradiene in the mutant SmKSL1: E550 + KR engineered bacteria increased by approximately 44 ​% compared to the wild type, the solubility of the protein increased by 24 ​%, and the catalytic efficiency (Kcat/Km) increased by 26 ​%. This paper conducted a preliminary study on the influence of the distal flexible region of the protein on the physicochemical properties of the enzyme, establishing critical molecular targets for the rational design of abietane diterpene synthases, while the high-performance mutants obtained provide superior enzymatic components for constructing biosynthesis platforms of pharmacologically relevant metabolites.